Interferon-β production modulating Listeria strains and methods for using same

ABSTRACT

Mutant  Listeria  bacteria that modulate interferon-B production are provided. The subject bacteria are characterized by having a mutation in a gene chosen from a TetR gene, a LadR gene, a VirR gene, a MarR gene a MdrL gene, a MdrT gene and a MdrM gene. The subject bacteria find use in a variety of applications, where representative applications of interest include, but are not limited to: (a) use of the subject bacteria as adjuvants; (b) use of the subject bacteria as delivery vectors for introducing macromolecules into a cell; (c) use of the subject bacteria as vaccines for eliciting or boosting a cellular immune response; etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of priorU.S. provisional application Ser. No. 60/860,982 filed Nov. 22, 2006,prior U.S. provisional application Ser. No. 60/923,375 filed Apr. 12,2007 and prior U.S. provisional application Ser. No. 60/963,230 filedAug. 3, 2007, disclosures of which applications are herein incorporatedby reference.

GOVERNMENT RIGHTS

This invention was made with a government support under federal grantno. AI063302 awarded by National Institutes of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Interferons (also referred to as “IFN” or “IFNs”) are proteins having avariety of biological activities, some of which are antiviral,immunomodulating and antiproliferative. They are relatively small,species-specific, single chain polypeptides, produced by mammalian cellsin response to exposure to a variety of inducers such as viruses,polypeptides, mitogens and the like. Interferons protect animal tissuesand cells against viral attack and are an important host defensemechanism. In most cases, interferons provide better protection totissues and cells of the kind from which they have been produced than toother types of tissues and cells, indicating that human-derivedinterferon could be more efficacious in treating human diseases thaninterferons from other species.

Interferons have potential in the treatment of a large number of humancancers since these molecules have anti-cancer activity which acts atmultiple levels. First, interferon proteins can directly inhibit theproliferation of human tumor cells. The anti-proliferative activity isalso synergistic with a variety of approved chemotherapeutic agents suchas CISPLATIN, 5FU and TAXOL. Secondly, the immunomodulatory activity ofinterferon proteins can lead to the induction of an anti-tumor immuneresponse. This response includes activation of NK cells, stimulation ofmacrophage activity and induction of MHC class I surface expressionleading to the induction of anti-tumor cytotoxic T lymphocyte activity.In addition, interferons also play a role in cross-presentation ofantigens in the immune system.

Moreover, some studies further indicate that IFN-β protein may haveanti-angiogenic activity. Angiogenesis, new blood vessel formation, iscritical for the growth of solid tumors. Evidence indicates that IFN-βmay inhibit angiogenesis by inhibiting the expression of pro-angiogenicfactors such as bFGF and VEGF. Lastly, interferon proteins may inhibittumor invasiveness by affecting the expression of enzymes such ascollagenase and elastase which are important in tissue remodeling.

Interferons also appear to have antiviral activities that are based ontwo different mechanisms. For instance, type I interferon proteins (αand β) can directly inhibit the replication of human hepatitis B virus(“HBV”) and hepatitis C virus (“HCV”), but can also stimulate an immuneresponse which attacks cells infected with these viruses.

The method of administering interferon is an important factor in theclinical application of this important therapeutic agent. Systemicadministration of interferon protein by either intravenous,intramuscular or subcutaneous injection has been most frequently usedwith some success in treating disorders such as hairy cell leukemia,Acquired Immune Deficiency Syndrome (AIDS) and related Kaposi's sarcoma.It is known, however, that proteins in their purified form areespecially susceptible to degradation. In particular, for interferon-β,the primary mechanism(s) of interferon degradation in solution areaggregation and deamidation. The lack of interferon stability insolutions and other products has heretofore limited its utility.Therefore, a more effective method of modulating the level ofinterferons, such as interferon-β, is needed.

SUMMARY OF THE INVENTION

Mutant Listeria bacteria that modulate interferon-β production areprovided. The subject bacteria are characterized by having a mutationwhich modulates the expression of a multidrug resistance transporter.The subject bacteria find use in a variety of applications, whererepresentative applications of interest include, but are not limited to:(a) use of the subject bacteria as adjuvants; (b) use of the subjectbacteria as delivery vectors for introducing macromolecules into a cell;(c) use of the subject bacteria as vaccines for eliciting or boosting acellular immune response; etc.

The present invention provides a Listeria bacterium having a mutationwhich modulates the expression of a multidrug resistance transporter,wherein the Listeria bacterium modulates interferon-β production inmacrophages. In some embodiments, the mutation is a mutation in atranscription regulator gene. In some embodiments, the transcriptionregulator gene is chosen from a TetR gene, a LadR gene, a VirR gene, anda MarR gene. In some embodiments, the mutation is a mutation in amultidrug resistance transporter gene. In some embodiments, themultidrug resistance transporter gene is chosen from a MdrL gene, a MdrTgene and a MdrM gene. In some embodiments, the mutation in thetranscription regulator or multidrug resistance transporter gene is aninsertion mutation. In some embodiments, the mutation in thetranscription regulator or multidrug resistance transporter gene is adeletion mutation. In certain embodiments, the Listeria bacterium isListeria monocytogenes. In further embodiments, the Listeria bacteriumis attenuated.

In some embodiments, the Listeria bacterium increases interferon-βproduction in macrophages as compared to a wild type Listeria bacterium.In other embodiments, the Listeria bacterium decreases interferon-βproduction in macrophages as compared to a wild type Listeria bacterium.

In some embodiments, the bacterium includes a heterologous nucleic acid.In further embodiments, the heterologous nucleic acid is integrated. Insome embodiments, the heterologous nucleic acid encodes at least oneproduct. In certain embodiments, the at least one product is an antigen.In some embodiments, the bacterium further includes a mutation in a UvrAgene and/or a UvrB gene.

In some embodiments, the Listeria bacterium induces an altered Type Iinterferon response in an infected subject as compared to a wild typeListeria bacterium. In further embodiments, the CD4+ and/or CD8+ T cellspecific response to Listeria epitopes is altered in the Listeriabacterium as compared to a wild type Listeria bacterium.

The present invention also provides a Listeria bacterium having amutation in a gene chosen from a TetR gene, a LadR gene, a VirR gene, aMarR gene, a MdrL gene, a MdrT gene and a MdrM gene. In someembodiments, the mutation is an insertion mutation. In some embodiments,the mutation is a deletion mutation. In certain embodiments, theListeria bacterium is Listeria monocytogenes. In further embodiments,the Listeria bacterium is attenuated.

In some embodiments, the bacterium includes a heterologous nucleic acid.In further embodiments, the heterologous nucleic acid is integrated. Insome embodiments, the heterologous nucleic acid encodes at least oneproduct. In certain embodiments, the at least one product is an antigen.In some embodiments, the bacterium further includes a mutation in a UvrAgene and/or a UvrB gene.

The present invention also provides a vaccine including attenuatedListeria bacteria having a mutation which modulates the expression of amultidrug resistance transporter, wherein the attenuated Listeriabacterium modulates interferon-β production in macrophages. In someembodiments, the mutation is a mutation in a transcription regulatorgene. In some embodiments, the transcription regulator gene is chosenfrom a TetR gene, a LadR gene, a VirR gene, and a MarR gene. In someembodiments, the mutation is a mutation in a multidrug resistancetransporter gene. In some embodiments, the multidrug resistancetransporter gene is chosen from a MdrL gene, a MdrT gene and a MdrMgene. In some embodiments, the mutation in the transcription regulatoror multidrug resistance transporter gene is an insertion mutation. Insome embodiments, the mutation in the transcription regulator ormultidrug resistance transporter gene is a deletion mutation. In certainembodiments, the Listeria bacterium is Listeria monocytogenes. In someembodiments, the bacterium further includes a mutation in a UvrA geneand/or a UvrB gene.

The present invention also provides a method of eliciting or boosting acellular immune response in a subject by administering to the subject aneffective amount of a vaccine including attenuated Listeria bacteriahaving a mutation which modulates the expression of a multidrugresistance transporter, wherein the attenuated Listeria bacteriummodulates interferon-β production in macrophages.

The present invention also provides a method for modulating interferon-βproduction in a human subject by administering to a human subject aneffective amount of an attenuated Listeria bacterium having a mutationwhich modulates the expression of a multidrug resistance transporter,wherein the Listeria bacterium modulates interferon-β production inmacrophages, wherein the administering modulates interferon-β productionin the human subject.

In some embodiments, the mutation is a mutation in a transcriptionregulator gene. In some embodiments, the transcription regulator gene ischosen from a TetR gene, a LadR gene, a VirR gene, and a MarR gene. Insome embodiments, the mutation is a mutation in a multidrug resistancetransporter gene. In some embodiments, the multidrug resistancetransporter gene is chosen from a MdrL gene, a MdrT gene and a MdrMgene. In some embodiments, the mutation in the transcription regulatoror multidrug resistance transporter gene is an insertion mutation. Insome embodiments, the mutation in the transcription regulator ormultidrug resistance transporter gene is a deletion mutation. In certainembodiments, the Listeria bacterium is Listeria monocytogenes. In someembodiments, the bacterium further includes a mutation in a UvrA geneand/or a UvrB gene. In some embodiments, the Listeria bacteriumincreases interferon-β production in macrophages as compared to a wildtype Listeria bacterium. In other embodiments, the Listeria bacteriumdecreases interferon-β production in macrophages as compared to a wildtype Listeria bacterium.

In some embodiments, the human subject has a neoplastic condition, suchas cancer. In some embodiments, the human subject has a viral infection,such as a Hepatitis C viral infection. In some embodiments, the humansubject has multiple sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 is a schematic showing an exemplary screen of Listeriamonocytogenes candidates to identify mutant bacteria that modulate IFN-βproduction in macrophages. Panel A shows candidate Listeriamonocytogenes mutants grown in 96 well plates overnight in BHI media.Panel B shows the addition of L. monocytogenes to macrophage culturesand incubation for a period of time to allow infection of themacrophages by the candidate mutant L. monocytogenes (T=0, infection ofthe macrophages; T=1 hour, macrophages are washed and gentamicin isadded; T=6 hours, supernatant is removed). Panel C shows testing of thesupernatants to identify candidate stains that modulate IFN-βproduction. ISRE cells are plated overnight and then the supernatantsfrom panel B are added and incubated for a period of time sufficient toreporter molecule detection. After the incubation, luciferase reagentsare added and the luminescence measured from each well to determinecultures of L. monocytogenes that modulate IFN-β production.

FIG. 2 is a graph showing the level of IFN-β secretion using theL-929-ISRE reporter assay for wild type L. monocytogenes and mutant L.monocytogenes strains that have a mutation in the LadR gene (DP-L5396),the VirR gene (DP-L5398), or the TetR gene (DP-L5397).

FIG. 3 is a graph showing the level of IFN-β induction as measuring byreal-time Q-PCR for wild type L. monocytogenes and mutant L.monocytogenes strains that have a mutation in the LadR gene (DP-L5396),the VirR gene (DP-L5398), or the TetR gene (DP-L5397).

FIG. 4 is a graph showing the intracellular growth of wild type L.monocytogenes and mutant L. monocytogenes strains that have a mutationin the LadR gene (DP-L5396)(□), the VirR gene (DP-L5398)(Δ), or the TetRgene (DP-L5397)(X).

FIG. 5 is a graph showing the expression level of the lmo2588 gene(multi drug transporter regulated by TetR) in wild type L. monocytogenesand the TetR mutant L. monocytogenes strain (DP-L5397).

FIG. 6 is a graph showing the expression level of the lmo1409 gene(multi drug transporter regulated by LadR) in wild type L. monocytogenesand the LadR mutant L. monocytogenes strain (DP-L5396).

FIG. 7. includes graphs and a schematic diagram showing that L.monocytogenes strains with mutations in regulators of multidrugresistance transporters induce altered host IFN-β responses. a.Intracellular growth curves of w.t. L. monocytogenes (▪), ladR::Tn917(▴), tetR::Tn917 (●), and marR::Tn917 (•), in bone marrow derivedmacrophages (BMM) (Portnoy et al., ibid). b. Schematic presentation ofsite of transposon insertions (marked with triangles), mapped to genespredicted to be transcription regulators. c. Quantitative RT-PCR(qRT-PCR) analysis of IFN-β gene induction in BMM in response toinfection with w.t. L. monocytogenes, ladR::Tn917, tetR::Tn917, andmarR::Tn917. d. Lactate dehydrogenase (LDH) release assay was performedon macrophages infected with w.t. L. monocytogenes, tetR::Tn917, marRand LadR mutants at various time points post infection. L. monocytogenescytotoxic LLO mutant S44A (Decatur, A. L. & Portnoy, D. A. A PEST-likesequence in listeriolysin O essential for Listeria monocytogenespathogenicity. Science 290, 992-5 (2000); Schnupf, P. et al. Regulatedtranslation of listeriolysin O controls virulence of Listeriamonocytogenes. Mol Microbiol 61, 999-1012 (2006)) was used as a positivecontrol. e. qRT-PCR analysis of L. monocytogenes MDR transportersexpression in w.t. bacteria, ladR-, tetR::Tn917, and marR-mutants grownto mid-log in BHI broth. f. qRT-PCR analysis of MDRs expression by w.t.L. monocytogenes in the presence of the toxic compoundstetraphenylphosphonium (TPP) or rhodamine 6G (R6G). All error bars inFIG. 7 represent one standard deviation; n=2 or 3.

FIG. 8: includes graphs showing the role of the L. monocytogenes MDRtransporters mdrL, mdrT, and mdrM in the induction of IFN-β frommacrophages. a. Intracellular growth curves of w.t. L. monocytogenes (●)and the deletion mutants: mdrL- (▪), mdrT- (x), and mdrM- (♦) in BMMs(Portnoy et al., ibid.). b. qRT-PCR analysis of IFN-β induction in BMMsinfected with w.t. L. monocytogenes, mdrL-, mdrT-, mdrM-, and acomplemented mdrM-strain expressing mdrM from the IPTG inducible vectorpLIV2 (Fischetti et al., ibid.) c. qRT-PCR analysis of IFN-β inductionin BMMs infected with ladR-, marR- or the double deletions ofladR-/mdrL- or marR/mdrM-. d. qRT-PCR analysis of mdrT expression levelin w.t. L. monocytogenes bacteria containing IPTG-inducible plasmidpLIV2::mdrT, and analysis of IFN-β induction by this strain in infectedBMMs. All error bars in FIG. 8 represent one standard deviation; n=2 or3.

FIG. 9: includes graphs showing that induction of IFN-β by ladR, tetR,and marR mutants is independent of MyD88/Trif and MAVS but dependent onIRF-3. qRT-PCR analysis of IFN-β induction in C57BL/6 w.t. vs.myd88/trif−/− BMMs (a.), and C57BL/6 w.t. vs. irf3−/− BMMs (O'Connell etal., ibid) (b.) infected with w.t. L. monocytogenes, and ladR, marR,tetR regulator mutants. All error bars in FIG. 9 represent one standarddeviation; n=2. c. qRT-PCR analysis of IFN-β induction in C57BL/6 BMMsvs. MAVS−/− BMMs (Sun, Q. et al. The specific and essential role of MAVSin antiviral innate immune responses. Immunity 24, 633-42 (2006))infected with w.t. L. monocytogenes, marR, tetR-Tn917 mutants, ortransfected with poly [I:C] as a positive control.

FIG. 10: includes a graph showing that L. monocytogenes MDR expressiondetermines the magnitude of the host immune response. Genes identified,by microarray analysis, as having lower expression in mdrM-infectedIFNαβR−/− macrophages (by SAM) or higher expression in tetR::Tn917infected IFNαβR−/− macrophages (by SAM and at least 4 fold higher thanWt), as compared to their expression in w.t. L. monocytogenes infectedIFNαβR−/− macrophages. All data is represented as fold induction overuninfected macrophages, and is the average of 2 experiments. The (*)symbol indicates that these values are significantly different frommacrophages infected with w.t. L. monocytogenes, by SAM analysis with afalse discovery rate of 10%.

FIG. 11: includes a graph showing liver and spleen colonization bymutant strains and induction of IFN-β in vivo by ladR, tetR, and marRtransposon mutants. a. C57BL/6 mice were infected with 1×10⁴ (0.1 LD50)of w.t., mdrM-, marR-, or tetR::Tn917 L. monocytogenes. Organs werecollected 48 hours post infection, and bacterial numbers are representedas colony forming units (cfu) per organ. b. Detection of Type I IFNlevels in serum of Balb/C mice infected intravenous (1×10⁴ bacteria)with Hank's Buffered Salt Solution (HBSS), w.t. L. monocytogenes,ladR::Tn917, tetR::Tn917, or marR::Tn917 for 24 hours. Units arepresented as relative light units (RLU), detected by luciferase reporterISRE-L929 cell line assay (Jiang et al., ibid). For each strain, n=5mice, and the median value is represented by a horizontal line.

FIG. 12: includes graphs showing the results of the 52 hour time course,indicating that tetR and pump1617(mdrM) mutants are slightly attenuatedat 24 h in the liver and 52 h in the spleen.

FIG. 13: includes a table and graphs indicating that tetR and ladRstrains show elevated Type I IFN responses relative to w.t. L.monocytogenes.

FIG. 14: includes graphs indicating that MCP-1, IFN-γ, IL-12p70, TNF-α,IL-10, and IL-6 secretion is lower for mutant than for w.t. L.monocytogenes strains.

FIG. 15: shows a schematic and conditions for the natural killer (NK)cell activation assay of FIG. 16.

FIG. 16: includes a graph indicating that all L. monocytogenes strains,mutant and w.t., yielded similar liver NK cell maturation profiles.

FIG. 17: includes a graph indicating that mdrM (pump1617) infectionresults in less NK activation than other mutant and w.t. L.monocytogenes strains.

FIG. 18: includes a table and graphs showing experimental conditions andresults indicating that MdrM (pump1617) infection induces a greater L.monocytogenes epitope-specific CD8+ T cell response than ladR and w.t.strains.

FIG. 19: includes a table and graphs showing experimental conditions andresults indicating that tetR infection induces a reduced L.monocytogenes epitope-specific CD8+ T cell response relative to othermutant and w.t. strains.

FIG. 20: includes a table and graphs showing experimental conditions andresults indicating that CD4+ T cell specific epitopic response wasreduced in the case of tetR and elevated relative to w.t. by mdrM(pump1617) infection.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

All references cited in the present disclosure are herein incorporatedby reference exactly as though each were individually incorporated byreference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the compound”includes reference to one or more compounds and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the presence of”includes determining the amount of something present, and/or determiningwhether it is present or absent. As used herein, the terms“determining,” “measuring,” and “assessing,” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

Overview

Mutant Listeria bacteria that modulate interferon-β production areprovided. The subject bacteria are characterized by having a mutation ina transcription regulator gene chosen from a TetR gene, a LadR gene, aVirR gene, and a MarR gene, or a mutation in a multidrug resistancetransporter gene chosen from a MdrL gene, a MdrT gene and a MdrM gene.The subject bacteria find use in a variety of applications, whererepresentative applications of interest include, but are not limited to:(a) use of the subject bacteria as adjuvants; (b) use of the subjectbacteria as delivery vectors for introducing macromolecules into a cell;(c) use of the subject bacteria as vaccines for eliciting or boosting acellular immune response; etc.

In further describing the subject invention, the subject mutant Listeriabacteria are reviewed first in greater detail, followed by a review ofrepresentative applications in which the subject vectors and methodsfind use.

Compositions

As noted above, the present invention provides mutant Listeria bacteriathat include a mutation which modulates the expression of a multidrugresistance transporter, wherein the Listeria bacteria modulateinterferon-β production, e.g., in macrophages. The term “modulates” asused here refers to an increase or a decrease in interferon-βproduction. In some embodiments, the modulation is an increase ordecrease of at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,or at least about 100% as compared to a Listeria bacteria that does notinclude the mutation.

In some embodiments, the mutant Listeria bacteria increase interferon-βproduction as compared to Listeria bacteria that do not include themutation, such as a wild type Listeria bacterium. In such embodiments,the increase is from about 1.5-fold increase to about 50-fold increaseor more, including about 2-fold increase to about 45-fold increase,about 5-fold increase to about 40-fold increase, about 10-fold increaseto about 35-fold increase, about 15-fold increase to about 30-foldincrease, about 20-fold increase to about 30-fold increase, and thelike.

In other embodiments, the mutant Listeria bacteria decrease interferon-βproduction as compared to Listeria bacteria that do not include themutation. In such embodiments, the increase is from about 1.5-folddecrease to about 50-fold decrease or more, including about 2-folddecrease to about 45-fold decrease, about 5-fold decrease to about40-fold decrease, about 10-fold decrease to about 35-fold decrease,about 15-fold decrease to about 30-fold decrease, about 20-fold decreaseto about 30-fold decrease, and the like.

In certain embodiments, mutant species according to the subjectinvention are ones that modulate (e.g., increase or decrease)interferon-β production as compared to their corresponding wild typestrain in a macrophage cell culture as described in the Experimentalsection, below. In this assay, macrophages are infected with test andreference, e.g., wild-type, strains of bacteria. Following a period oftime, e.g., 4 to 18 hours, the macrophage culture media is collected andthe amount of Type I interferon secreted by the macrophages is detectedusing a reporter gene such as luciferase cloned under regulation of aType I interferon signaling pathway. The level of the reporter gene isthen measured test and reference, e.g., wild-type, strains of bacteriato identify mutant Listeria strains that modulate (e.g., increase ordecrease) interferon-β production.

A “transcription regulator” as used herein typically refers to a protein(or polypeptide) which affects the transcription, and accordingly theexpression, of a specified gene. A transcription regulator may refer toa single polypeptide transcription factor, one or more polypeptidesacting sequentially or in concert, or a complex of polypeptides. Inaddition, a transcription regulator may affect the transcription, andaccording the expression, or a gene by directly increasing or decreasingtranscription of a target gene, or by indirectly increasing ordecreasing transcription of a first gene as part of cascade that thenaffects the transcription of the target gene.

A “multidrug resistance transporter” as used herein refers to a protein(or polypeptide) which participates in conferring to a cell resistanceto multiple cytotoxic insults. A multidrug resistance transporter mayrefer to a single polypeptide, one or more polypeptides actingsequentially or in concert, or a complex of polypeptides. In addition, amultidrug resistance transporter may participate, directly orindirectly, in the catalysis of energy-dependent extrusion of moleculesor compounds out of a cell or their partitioning into a specificintracellular compartment.

A “gene” as used in the context of the present invention is a sequenceof nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.)with which a genetic function is associated. A gene is a hereditaryunit, for example of an organism, comprising a polynucleotide sequence(e.g., a DNA sequence for mammals) that occupies a specific physicallocation (a “gene locus” or “genetic locus”) within the genome of anorganism. A gene can encode an expressed product, such as a polypeptideor a polynucleotide (e.g., tRNA). Typically, a gene includes codingsequences, such as, polypeptide encoding sequences, and non-codingsequences, such as, promoter sequences, and transcriptional regulatorysequences (e.g., enhancer sequences). In certain cases, a gene may sharesequences with another gene(s) (e.g., overlapping genes).

Exemplary transcription regulator genes include, but are not limited to,TetR (lmo2589) as described in greater detail in Ramos et al., MicrobiolMol Biol Rev. 2005 June; 69(2): 326-356; LadR (lmo1408) as described ingreater detail in Huillet et al., FEMS Microbiol Lett. 2006 January;254(1):87-94.; VirR (lmo1745) as described in greater detail in Mandinet al., Mol Microbiol. 2005 September; 57(5):1367-80.; and MarR(lmo1618) as described in greater detail in Martin et al., Proc NatlAcad Sci USA. 1995 Jun. 6; 92(12):5456-60.

Exemplary multidrug resistance transporter genes include, but are notlimited, to members of the major facilitator superfamily including MdrL(lmo1409) as described in greater detail in Mata et al., FEMS Microbiol.Lett. 2000 Jun. 15; 187(2):185-8; MdrT (lmo2588) and MdrM (lmo1617), asdescribed herein.

The subject bacteria may be any Listeria species that includes amutation according to the subject invention. Thus, strains of Listeriaother than L. monocytogenes may be used for the generation of mutantsaccording to the present invention. In certain embodiments, the Listeriastrain is L. monocytogenes.

Specific mutant Listeria bacteria of interest that include a mutatedtranscription regulator gene include, but are not limited to: DP-L5397,DP-L5396, DP-L5398, DP-L5418, DP-L5441 and DP-L5442, where thesespecific strains are described below in greater detail. DP-L5397,DP-L5396, DP-L5398, DP-L5418, DP-L5441 and DP-L5442 are deposited withthe American Type Culture Collection depository (10801 UniversityBoulevard, Manassas, Va. 20110-2209).

Specific mutant Listeria bacteria of interest that include a mutatedmultidrug resistance transporter gene include, but are not limited to:DP-L5449, DP-L5443, DP-L5448, DP-L5444 and DP-L5445, where thesespecific strains are described below in greater detail. DP-L5449,DP-L5443, DP-L5448, DP-L5444 and DP- L5445 are deposited with theAmerican Type Culture Collection depository (10801 University Boulevard,Manassas, Va. 20110-2209).

Specific mutant Listeria bacteria of interest that include both amutated transcription regulator gene and a mutated multidrug resistancetransporter gene include, but are not limited to: DP-L5446, andDP-L5447, where these specific strains are described below in greaterdetail. DP-L5446, and DP-L5447 are deposited with the American TypeCulture Collection depository (10801 University Boulevard, Manassas, Va.20110-2209).

The above-mutant bacteria may be fabricated using a variety of differentprotocols. As such, generation of the subject mutant bacteria may beaccomplished in a number of ways that are well known to those of skillin the art, including deletion mutagenesis, insertion mutagenesis, andmutagenesis which results in the generation of frameshift mutations,mutations which effect premature termination of a protein, or mutationof regulatory sequences which affect gene expression. Mutagenesis can beaccomplished using recombinant DNA techniques or using traditionalmutagenesis technology using mutagenic chemicals or radiation andsubsequent selection of mutants. Representative protocols of differentways to generate mutant bacteria according to the present invention areprovided in the Experimental Section, below.

In certain embodiments, the mutant Listeria bacteria are killed butmetabolically active (KBMA). By the term “KBMA” or “killed butmetabolically active” is meant that the bacteria are attenuated forentry into non-phagocytic cells and attenuated with respect tocell-to-cell spread resulting in bacteria that have greatly reducedtoxicity and yet the immunogenicity of the bacteria is maintained. Suchmutants include, but are not limited to, mutations in one or all uvrgenes, i.e. uvrA, uvrB, uvrC, and uvrD genes as well as recA genes, orfunctionally equivalent genes, depending on the genus and species of themicrobe. These mutations result in attenuation in the activity of thecorresponding enzymes UvrA (an ATPase), UvrB (a helicase), UvrC (anuclease), UvrD (a helicase II) and RecA (a recombinase). These mutantswould typically be used in conjunction with a crosslinking compound,such as a psoralen. In one embodiment, there are attenuating mutations,such as deletions, in both uvrA and uvrB (uvrAB). KBMA mutations arefurther described in Brockstedt et al., Nature Med. 11, 853-860 (2005)and in U.S. Published Patent Application No. 2004/0228877.

In certain embodiments, the mutant Listeria bacteria are alsoattenuated. By the term “attenuation,” as used herein, is meant adiminution in the ability of the bacterium to cause disease in ananimal. In other words, the pathogenic characteristics of the attenuatedListeria strain have been lessened compared with wild-type Listeria,although the attenuated Listeria is capable of growth and maintenance inculture. Using as an example the intravenous inoculation of Balb/c micewith an attenuated Listeria, the lethal dose at which 50% of inoculatedanimals survive (LD₅₀) is preferably increased above the LD₅₀ ofwild-type Listeria by at least about 10-fold, more preferably by atleast about 100-fold, more preferably at least about 1,000 fold, evenmore preferably at least about 10,000 fold, and most preferably at leastabout 100,000-fold. An attenuated strain of Listeria is thus one whichdoes not kill an animal to which it is administered, or is one whichkills the animal only when the number of bacteria administered is vastlygreater than the number of wild type non-attenuated bacteria which wouldbe required to kill the same animal. An attenuated bacterium should alsobe construed to mean one which is incapable of replication in thegeneral environment because the nutrient required for its growth is notpresent therein. Thus, the bacterium is limited to replication in acontrolled environment wherein the required nutrient is provided. Theattenuated strains of the present invention are thereforeenvironmentally safe in that they are incapable of uncontrolledreplication.

In certain embodiments, the attenuated mutant Listeria bacteriaaccording to the subject invention are ones that exhibit a decreasedvirulence compared to their corresponding wild type strain in theCompetitive Index Assay as described in Auerbach et al., “Development ofa Competitive Index Assay To Evaluate the Virulence of Listeriamonocytogenes actA Mutants during Primary and Secondary Infection ofMice,” Infection and Immunity, September 2001, p. 5953-5957, Vol. 69,No. 9. In this assay, mice are inoculated with test and reference, e.g.,wild-type, strains of bacteria. Following a period of time, e.g., 48 to60 hours, the inoculated mice are sacrificed and one or more organs,e.g., liver, spleen, are evaluated for bacterial abundance. In theseembodiments, a given bacterial strain is considered to be less virulentif its abundance in the spleen is at least about 50-fold, or more, suchas 70-fold or more less than that observed with the correspondingwild-type strain, and/or its abundance in the liver is at least about10-fold less, or more, such as 20-fold or more less than that observedwith the corresponding wild-type strain.

In yet other embodiments, bacteria are considered to be less virulent ifthey show abortive replication in less than about 8 hours, such as lessthan about 6 hours, including less than about 4 hours, as determinedusing the assay described in Jones and Portnoy, Intracellular growth ofbacteria. (1994b) Methods Enzymol. 236:463-467. In yet otherembodiments, bacteria are considered to be attenuated or less virulentif, compared to wild-type, they form smaller plaques in the plaque assayemployed in the Experimental Section, below, where cells, such as murineL2 cells, are grown to confluency, e.g., in six-well tissue culturedishes, and then infected with bacteria. Subsequently, DME-agarcontaining gentamicin is added and plaques are grown for a period oftime, e.g., 3 days. Living cells are then visualized by adding anadditional DME-agar overlay, e.g., containing neutral red (GIBCO BRL)and incubated overnight. In such an assay, the magnitude in reduction inplaque size observed with the attenuated mutant as compared to thewild-type is, in certain embodiments, 10%, including 15%, such as 25% ormore.

In certain embodiments, the subject bacteria are cytotoxic. A particularstrain of bacteria is considered to be cytotoxic if it compromises itshost cell in a period of less than about 8 hours, sometimes less thanabout 6 hours, e.g., in less than about 5 hours, less than about 4hours, less than about 3 hours, less than about two hours, or less thanabout 1 hour, as determined using the cytotoxicity assay describedbelow.

In certain embodiments, mutant bacteria according to the subjectinvention express a heterologous antigen. The heterologous antigen is,in certain embodiments, one that is capable of providing protection inan animal against challenge by the infectious agent from which theheterologous antigen was derived, or which is capable of affecting tumorgrowth and metastasis in a manner which is of benefit to a hostorganism. Heterologous antigens which may be introduced into a Listeriastrain of the subject invention by way of DNA encoding the same thusinclude any antigen which when expressed by Listeria serves to elicit acellular immune response which is of benefit to the host in which theresponse is induced. Heterologous antigens therefore include thosespecified by infectious agents, wherein an immune response directedagainst the antigen serves to prevent or treat disease caused by theagent. Such heterologous antigens include, but are not limited to,viral, bacterial, fungal or parasite surface proteins and any otherproteins, glycoproteins, lipoprotein, glycolipids, and the like.Heterologous antigens also include those which provide benefit to a hostorganism which is at risk for acquiring or which is diagnosed as havinga tumor that expresses the said heterologous antigen(s). The hostorganism is preferably a mammal and most preferably, is a human.

By the term “heterologous antigen,” as used herein, is meant a proteinor peptide, a lipoprotein or lipopeptide, or any other macromoleculewhich is not normally expressed in Listeria, which substantiallycorresponds to the same antigen in an infectious agent, a tumor cell ora tumor-related protein. The heterologous antigen is expressed by astrain of Listeria according to the subject invention, and is processedand presented to cytotoxic T-cells upon infection of mammalian cells bythe strain. The heterologous antigen expressed by Listeria species neednot precisely match the corresponding unmodified antigen or protein inthe tumor cell or infectious agent so long as it results in a T-cellresponse that recognizes the unmodified antigen or protein which isnaturally expressed in the mammal. In other examples, the tumor cellantigen may be a mutant form of that which is naturally expressed in themammal, and the antigen expressed by the Listeria species will conformto that tumor cell mutated antigen. By the term “tumor-related antigen,”as used herein, is meant an antigen which affects tumor growth ormetastasis in a host organism. The tumor-related antigen may be anantigen expressed by a tumor cell, or it may be an antigen which isexpressed by a non-tumor cell, but which when so expressed, promotes thegrowth or metastasis of tumor cells. The types of tumor antigens andtumor-related antigens which may be introduced into Listeria by way ofincorporating DNA encoding the same, include any known or heretoforeunknown tumor antigen. In other examples, the “tumor-related antigen”has no effect on tumor growth or metastasis, but is used as a componentof the Listeria vaccine because it is expressed specifically in thetissue (and tumor) from which the tumor is derived. In still otherexamples, the “tumor-related antigen” has no effect on tumor growth ormetastasis, but is used as a component of the Listeria vaccine becauseit is selectively expressed in the tumor cell and not in any othernormal tissues.

The heterologous antigen useful in vaccine development may be selectedusing knowledge available to the skilled artisan, and many antigenicproteins which are expressed by tumor cells or which affect tumor growthor metastasis or which are expressed by infectious agents are currentlyknown. For example, viral antigens which may be considered as useful asheterologous antigens include but are not limited to the nucleoprotein(NP) of influenza virus and the gag protein of HIV. Other heterologousantigens include, but are not limited to, HIV env protein or itscomponent parts gp120 and gp41, HIV nef protein, and the HIV polproteins, reverse transcriptase and protease. Still other heterologousantigens can be those related to hepatitis C virus (HCV), including butnot limited to the E1 and E2 glycoproteins, as well as non-structural(NS) proteins, for example NS3. In addition, other viral antigens suchas herpesvirus proteins may be useful. The heterologous antigens neednot be limited to being of viral origin. Parasitic antigens, such as,for example, malarial antigens, are included, as are fungal antigens,bacterial antigens and tumor antigens.

As noted herein, a number of proteins expressed by tumor cells are alsoknown and are of interest as heterologous antigens which may be insertedinto the vaccine strain of the invention. These include, but are notlimited to, the bcr/abl antigen in leukemia, HPVE6 and E7 antigens ofthe oncogenic virus associated with cervical cancer, the MAGE1 and MZ2-Eantigens in or associated with melanoma, and the MVC-1 and HER-2antigens in or associated with breast cancer. Other coding sequences ofinterest include, but are not limited to, costimulatory molecules,immunoregulatory molecules, and the like.

The introduction of DNA encoding a heterologous antigen into a strain ofListeria may be accomplished, for example, by the creation of arecombinant Listeria in which DNA encoding the heterologous antigen isharbored on a vector, such as a plasmid for example, which plasmid ismaintained and expressed in the Listeria species, and in whose antigenexpression is under the control of prokaryotic promoter/regulatorysequences. Alternatively, DNA encoding the heterologous antigen may bestably integrated into the Listeria chromosome by employing, forexample, transposon mutagenesis, homologous recombination, or integrasemediated site-specific integration (as described in U.S. patentapplication Ser. No. 10/136,860, the disclosure of which is hereinincorporated by reference).

Several approaches may be employed to express the heterologous antigenin Listeria species as will be understood by one skilled in the art oncearmed with the present disclosure. In certain embodiments, genesencoding heterologous antigens are designed to either facilitatesecretion of the heterologous antigen from the bacterium or tofacilitate expression of the heterologous antigen on the Listeria cellsurface.

In certain embodiments, a fusion protein which includes the desiredheterologous antigen and a secreted or cell surface protein of Listeriais employed. Listerial proteins which are suitable components of suchfusion proteins include, but are not limited to, ActA, listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase (PI-PLC). A fusionprotein may be generated by ligating the genes which encode each of thecomponents of the desired fusion protein, such that both genes are inframe with each other. Thus, expression of the ligated genes results ina protein comprising both the heterologous antigen and the Listerialprotein. Expression of the ligated genes may be placed under thetranscriptional control of a Listerial promoter/regulatory sequence suchthat expression of the gene is effected during growth and replication ofthe organism. Signal sequences for cell surface expression and/orsecretion of the fused protein may also be added to genes encodingheterologous antigens in order to effect cell surface expression and/orsecretion of the fused protein. When the heterologous antigen is usedalone (i.e., in the absence of fused Listeria sequences), it may beadvantageous to fuse thereto signal sequences for cell surfaceexpression and/or secretion of the heterologous antigen. The proceduresfor accomplishing this are well know in the art of bacteriology andmolecular biology.

The DNA encoding the heterologous antigen which is expressed is, in manyembodiments, preceded by a suitable promoter to facilitate suchexpression. The appropriate promoter/regulatory and signal sequences tobe used will depend on the type of Listerialprotein desired in thefusion protein and will be readily apparent to those skilled in the artof Listeria molecular biology. For example, suitable L. monocytogenespromoter/regulatory and/or signal sequences which may be used to directexpression of a fusion protein include, but are not limited to,sequences derived from the Listeria hly gene which encodes LLO, theListeria p60 (iap) gene, and the Listeria actA gene which encodes asurface protein necessary for L. monocytogenes actin assembly. Otherpromoter sequences of interest include the plcA gene which encodesPI-PLC, the Listeria mpl gene, which encodes a metalloprotease, and theListeria inlA gene which encodes internalin, a Listeria membraneprotein. The heterologous regulatory elements such as promoters derivedfrom phage and promoters or signal sequences derived from otherbacterial species may be employed for the expression of a heterologousantigen by the Listeria species.

In certain embodiments, the mutant Listeria include a vector. The vectormay include DNA encoding a heterologous antigen. Typically, the vectoris a plasmid that is capable of replication in Listeria. The vector mayencode a heterologous antigen, wherein expression of the antigen isunder the control of eukaryotic promoter/regulatory sequences, e.g., ispresent in an expression cassette. Typical plasmids having suitablepromoters that are of interest include, but are not limited to, pCMV-βcomprising the immediate early promoter/enhancer region of humancytomegalovirus, and those which include the SV40 early promoter regionor the mouse mammary tumor virus LTR promoter region.

As such, in certain embodiments, the subject bacteria include at leastone coding sequence for heterologous polypeptide/protein, as describedabove. In many embodiments, this coding sequence is part of anexpression cassette, which provides for expression of the codingsequence in the Listeria cell for which the vector is designed. The term“expression cassette” as used herein refers to an expression module orexpression construct made up of a recombinant DNA molecule containing atleast one desired coding sequence and appropriate nucleic acid sequencesnecessary for the expression of the operably linked coding sequence in aparticular host organism, i.e., the Listeria cell for which the vectoris designed, such as the promoter/regulatory/signal sequences identifiedabove, where the expression cassette may include coding sequences fortwo or more different polypeptides, or multiple copies of the samecoding sequence, as desired. The size of the coding sequence and/orexpression cassette that includes the same may vary, but typically fallswithin the range of about 25-30 to about 6000 bp, usually from about 50to about 2000 bp. As such, the size of the encoded product may varygreatly, and a broad spectrum of different products may be encoded bythe expression cassettes present in the vectors of this embodiment.

As indicated above, the vector may include at least one coding sequence,where in certain embodiments the vectors include two or more codingsequences, where the coding sequences may encode products that actconcurrently to provide a desired results. In general, the codingsequence may encode any of a number of different products and may be ofa variety of different sizes, where the above discussion merely providesrepresentative coding sequences of interest.

Adjuvant Compositions

The subject mutant bacterial strains also find use as immunopotentiatingagents, i.e., as adjuvants. In such applications, the subject attenuatedbacteria may be administered in conjunction with an immunogen, e.g., atumor antigen, modified tumor cell, etc., according to methods known inthe art where live bacterial strains are employed as adjuvants. See,e.g., Berd et al., Vaccine 2001 Mar. 21; 19(17-19):2565-70.

In some embodiments, the mutant bacterial strains are employed asadjuvants by chemically coupled to a sensitizing antigen. Thesensitizing antigen can be any antigen of interest, where representativeantigens of interest include, but are not limited to: viral agents,e.g., Herpes simplex virus; malaria parasite; bacteria, e.g.,staphylococcus aureus bacteria, diphtheria toxoid, tetanus toxoid,shistosomula; tumor cells, e.g. CAD₂ mammary adenocarcinomia tumorcells, and hormones such as thyroxine T₄, triiiodothyronine T₃, andcortisol. The coupling of the sensitizing antigen to theimmunopotentiating agent can be accomplished by means of variouschemical agents having two reactive sites such as, for example,bisdiazobenzidine, glutaraldehyde, di-iodoacetate, and diisocyanates,e.g., m-xylenediisocyanate and toluene-2.4-diisocyanate. Use of Listeriaspp. as adjuvants is further described in U.S. Pat. No. 4,816,253.

Vaccines

The subject attenuated mutant bacteria also find use as vaccines. Thevaccines of the present invention are administered to a vertebrate bycontacting the vertebrate with a sublethal dose of an attenuated mutantListeria vaccine, where contact typically includes administering thevaccine to the host. In many embodiments, the attenuated bacteria areprovided in a pharmaceutically acceptable formulation. Administrationcan be oral, parenteral, intranasal, intramuscular, intradermal,intraperitoneal, intravascular, subcutaneous, direct vaccination oflymph nodes, administration by catheter or any one or more of a varietyof well-known administration routes. In farm animals, for example, thevaccine may be administered orally by incorporation of the vaccine infeed or liquid (such as water). It may be supplied as a lyophilizedpowder, as a frozen formulation or as a component of a capsule, or anyother convenient, pharmaceutically acceptable formulation that preservesthe antigenicity of the vaccine. Any one of a number of well knownpharmaceutically acceptable diluents or excipients may be employed inthe vaccines of the invention. Suitable diluents include, for example,sterile, distilled water, saline, phosphate buffered solution, and thelike. The amount of the diluent may vary widely, as those skilled in theart will recognize. Suitable excipients are also well known to thoseskilled in the art and may be selected, for example, from A. Wade and P.J. Weller, eds., Handbook of Pharmaceutical Excipients (1994) ThePharmaceutical Press: London. The dosage administered may be dependentupon the age, health and weight of the patient, the type of patient, andthe existence of concurrent treatment, if any. The vaccines can beemployed in dosage forms such as capsules, liquid solutions,suspensions, or elixirs, for oral administration, or sterile liquid forformulations such as solutions or suspensions for parenteral, intranasalintramuscular, or intravascular use. In accordance with the invention,the vaccine may be employed, in combination with a pharmaceuticallyacceptable diluent, as a vaccine composition, useful in immunizing apatient against infection from a selected organism or virus or withrespect to a tumor, etc. Immunizing a patient means providing thepatient with at least some degree of therapeutic or prophylacticimmunity against selected pathogens, cancerous cells, etc.

The subject vaccines find use in methods for eliciting or boosting acellular immune response, e.g., a helper T cell or a cytotoxic T-cellresponse to a selected agent, e.g., pathogenic organism, tumor, etc., ina vertebrate, where such methods include administering an effectiveamount of the Listeria vaccine. The subject vaccines find use in methodsfor eliciting in a vertebrate an innate immune response that augmentsthe antigen-specific immune response. Furthermore, the vaccines of thepresent invention may be used for treatment post-exposure or postdiagnosis. In general, the use of vaccines for post-exposure treatmentwould be recognized by one skilled in the art, for example, in thetreatment of rabies and tetanus. The same vaccine of the presentinvention may be used, for example, both for immunization and to boostimmunity after exposure. Alternatively, a different vaccine of thepresent invention may be used for post-exposure treatment, for example,such as one that is specific for antigens expressed in later stages ofexposure. As such, the subject vaccines prepared with the subjectvectors find use as both prophylactic and therapeutic vaccines to induceimmune responses that are specific for antigens that are relevant tovarious disease conditions.

The patient may be any human and non-human animal susceptible toinfection with the selected organism. The subject vaccines will findparticular use with vertebrates such as man, and with domestic animals.Domestic animals include domestic fowl, bovine, porcine, ovine, equine,caprine, Leporidate (such as rabbits), or other animal which may be heldin captivity.

In general, the subject vaccines find use in vaccination applications asdescribed U.S. Pat. Nos. 5,830,702 and 6,051,237, as well as PCTpublication no WO 99/25376.

Methods

The present invention also provides methods for modulating interferon-βproduction in a subject, by administering to a subject an effectiveamount of an attenuated Listeria bacterium comprising a mutation in atranscription regulator gene and/or multidrug resistance transportergene, wherein the attenuated Listeria bacterium modulates interferon-βproduction in macrophages, and wherein the administering modulatesinterferon-β production in the subject.

As used herein “therapeutically effective amount” or “efficaciousamount” means the amount of an organism or compound that, whenadministered to a mammal or other subject for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the organismor compound, the disease and its severity and the age, weight, etc., ofthe subject to be treated.

In some embodiments, subjects suitable for treatment with a method ofthe present invention include individuals having a cellularproliferative disease, such as a neoplastic disease (e.g., cancer).Cellular proliferative disease is characterized by the undesiredpropagation of cells, including, but not limited to, neoplastic diseaseconditions, e.g., cancer. Examples of cellular proliferative diseaseinclude, but not limited to, abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid tumors and tumor metastasis, benigntumors, for example, hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders, Bechet's disease, goutor gouty arthritis, abnormal angiogenesis accompanying, for example,rheumatoid arthritis, psoriasis, diabetic retinopathy, other ocularangiogenic diseases such as retinopathy of prematurity (retrolentalfibroplastic), macular degeneration, corneal graft rejection,neurovascular glaucoma and Oster Webber syndrome, psoriasis, restenosis,fungal, parasitic and viral infections such cytomegaloviral infections.Subjects to be treated according to the methods of the invention includeany individual having any of the above-mentioned disorders.

In other embodiments, subjects suitable for treatment with a method ofthe present invention include individuals who have been clinicallydiagnosed as infected with a hepatitis virus (e.g., HAV, HBV, HCV,delta, etc.), particularly HCV, are suitable for treatment with themethods of the instant invention. Individuals who are infected with HCVare identified as having HCV RNA in their blood, and/or having anti-HCVantibody in their serum. Such individuals include naïve individuals(e.g., individuals not previously treated for HCV, particularly thosewho have not previously received IFN-α-based or ribavirin-based therapy)and individuals who have failed prior treatment for HCV.

In other embodiments, subjects suitable for treatment with a method ofthe present invention include individuals having multiple sclerosis.Multiple sclerosis refers to an autoimmune neurodegenerative disease,which is marked by inflammation within the central nervous system withlymphocyte attack against myelin produced by oligodendrocytes, plaqueformation and demyelization with destruction of the myelin sheath ofaxons in the brain and spinal cord, leading to significant neurologicaldisability over time. Typically, at onset an otherwise healthy personpresents with the acute or sub acute onset of neurologicalsymptomatology (attack) manifested by unilateral loss of vision,vertigo, ataxia, dyscoordination, gait difficulties, sensory impairmentcharacterized by paresthesia, dysesthesia, sensory loss, urinarydisturbances until incontinence, diplopia, dysarthria or various degreesof motor weakness until paralysis. The symptoms are usually painless,remain for several days to a few weeks, and then partially or completelyresolve. After a period of remission, a second attack will occur. Duringthis period after the first attack, the patient is defined to sufferfrom probable MS. Probable MS patients may remain undiagnosed for years.When the second attack occurs the diagnosis of clinically definite MS(CDMS) is made (Poser criteria 1983; C. M. Poser et al., Ann. Neurol.1983; 13, 227).

The terms “subject” and “patient” mean a member or members of anymammalian or non-mammalian species that may have a need for thepharmaceutical methods, compositions and treatments described herein.Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. Humans and non-humananimals having commercial importance (e.g., livestock and domesticatedanimals) are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms. As used herein, the term “treating” is thus usedto refer to both prevention of disease, and treatment of pre-existingconditions. For example, where the mutant bacteria is administered, theprevention of cellular proliferation can be accomplished byadministration of the subject compounds prior to development of overtdisease, e.g. to prevent the regrowth of tumors, prevent metastaticgrowth, etc. Alternatively the compounds are used to treat ongoingdisease, by stabilizing or improving the clinical symptoms of thepatient.

Combination Therapy

For use in the subject methods, the subject mutant Listeria may beadministered in combination with other pharmaceutically active agents,including other agents that treat the underlying condition or a symptomof the condition. In addition, the mutant Listeria may be used toprovide an increase in the effectiveness of another chemical, such as apharmaceutical, that is necessary to produce the desired biologicaleffect.

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

Examples of other agents for use in combination therapy of neoplasticdisease include, but are not limited to, thalidomide, MARIMASTAT, COL-3,BMS-275291, squalamine, 2-ME, SU6668, NEOVASTAT, Medi-522, EMD121974,CAI, CELECOXIB interleukin-12, IM862, TNP470, AVASTIN, GLEEVAC,HERCEPTIN, and mixtures thereof. Examples of chemotherapeutic agents foruse in combination therapy include, but are not limited to,DAUNORUBICIN, daunomycin, dactinomycin, DOXORUBICIN, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithrarnycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelarnine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, TAXOL,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, CISPLATIN and diethylstilbestrol(DES).

Other antiviral agents can also be delivered in the treatment methods ofthe invention. For example, compounds that inhibit inosine monophosphatedehydrogenase (IMPDH) may have the potential to exert direct anti viralactivity, and such compounds can be administered in combination with themutant Listeria, as described herein. Drugs that are effectiveinhibitors of hepatitis C NS3 protease may be administered incombination with the mutant Listeria, as described herein. Hepatitis CNS3 protease inhibitors inhibit viral replication. Other agents such asinhibitors of HCV NS3 helicase are also attractive drugs forcombinational therapy, and are contemplated for use in combinationtherapies described herein. Ribozymes such as Heptazyme™ andphosphorothioate oligonucleotides which are complementary to HCV proteinsequences and which inhibit the expression of viral core proteins arealso suitable for use in combination therapies described herein.

Examples of other agents for use in combination therapy of multiplesclerosis include, but are not limited to; glatiramer; corticosteroids;muscle relaxants, such as Tizanidine (ZANAFLEX) and baclofen (LIORESAL);medications to reduce fatigue, such as amantadine (SYMMETREL) ormodafinil (PROVIGIL); and other medications that may also be used fordepression, pain and bladder or bowel control problems that can beassociated with MS.

In the context of a combination therapy, combination therapy compoundsmay be administered by the same route of administration (e.g.intrapulmonary, oral, enteral, etc.) that the mutant Listeria areadministered. In the alternative, the compounds for use in combinationtherapy with the mutant Listeria may be administered by a differentroute of administration.

Kits

Kits with unit doses of the subject mutant Listeria, e.g., in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the mutant Listeriain treating a pathological condition of interest.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Generation of Listeria monocytogenes Transcription RegulatorGene Mutants and Characterization of Role in Modulating Interferon-βExpression

Mutant Libraries

Generation of Listeria monocytogenes mutant libraries using transposonTn917-LTV3 was previously described (Camilli et al., 1990). Nineindependent libraries were generated by A. Camilli, from which 8libraries were screened in this study. These libraries are designated asLibraries 910-918 and are part of the Portnoy culture collection.

Genetic Screen

Eight independent L. monocytogenes Tn917-LTV3-transposon insertionlibraries, were plated to single colonies on BHI agar plates and thenpatched (single colony per well) to 96 well plates (FIG. 1, panel A). Atotal of 4500 bacterial insertion mutants were grown on BHI media in 96well plates and were frozen with glycerol for further infectionexperiments. Bone marrow-derived macrophages from C57B1/6 mice femurswere prepared as described and grown for six days and frozen (Portnoy etal., 1989). Macrophages were thawed and were plated on 96 well plates,4×10⁴ cells/well and were infected with 1×10⁶ of over-night grownbacterial cultures (FIG. 1, panel B). Half an hour post infection,macrophages were washed to remove extracellular bacteria and gentamicinwas added at a concentration of 50 μg/ml to prevent extracellular growthof bacteria. At 6 hours post infection 100 μl of macrophages culturemedia was taken and frozen at −80. The amount of Type I IFN secreted bymacrophages during bacterial infection was detected using the reportergene luciferase, cloned under the regulation of Type I interferonsignaling pathway in L929 cell line, ISRE-L929 (Jiang et al., 2005).ISRE-L929 cells were grown in 96 well plates and were incubated with 40μl of infected macrophages culture media for 4 h (FIG. 1, panel C).Then, cells were lysed and luciferase activity was detected using BrightGlow Assay (Promega, E-2620) and light emission measurement byluminescence counter (VICTOR3, PerkinElmer®) (FIG. 2). Screening ofmutants was done in triplicate for each mutant on the same plate and 8replicates of wild type L. monocytogenes and hly-minus mutant ascontrols. Table 1 shows the strains characterized and used in thisstudy, and FIG. 2 shows that the LadR, VirR, and TetR mutant Listeriastrains had an increase in interferon-β expression over the wild typestrain.

TABLE 1 Gene Portnoy lab No. name Description strain number 1 lmo2589TetR negative regulator DP-L5397 2 lmo1408 LadR negative regulatorDP-L5396 3 lmo1745 VirR transcription regulator DP-L5398 4 lmo1618 MarRtranscription regulator DP-L5442 5 lmo2588 Multi drug transporterregulated by DP-L5416 TetR (lmo2589) 6 lmo1617 Multi drug transporterregulated by DP-L5417 MarR (lmo1618) 7 lmo1409 Multi drug transporterregulated by DP-L5445 LadR (mdrL) (lmo1409)Analysis of Mutants

Analysis of IFN-β Transcription by Quantitative Real-Time PCR:

This analysis was performed as described by Auerbuch et al (Auerbuch V.et al., J Exp Med. 2004 Aug. 16; 200(4):527-33. Epub 2004 Aug. 9). Forfurther verification of induction of IFN-β by bacterial mutants, thelevel of IFN-β mRNA was analyzed and compared to induction of IFN-β bywild type L monocytogenes. Four hours post infection, total macrophagesRNA was extracted using RNeasy kit (QIAGENE™). First strand cDNA wasproduced by using 1 μg of total RNA in M-MLV reverse transcriptasereaction. SYBR® Green-based quantitative PCR amplification was usingSYBR® Green PCR core reagents (Applied Biosystems) and the Stratagene™Mx3000P Real-Time PCR System. Primers used: ifnb-F:5′-ctggagcagctgaatggaaag (SEQ ID NO:01); ifnb-R:5′-cttgaagtccgccctgtaggt (SEQ ID NO:02); β-actin-F:5′-aggtgtgatggtgggaatgg (SEQ ID NO:03); β-actin-R:5′-gcctcgtcacccacatagga (SEQ ID NO:04). FIG. 3 shows that the LadR,VirR, and TetR mutant Listeria strains induced an increase ininterferon-β mRNA expression over the wild type strain.

Intracellular Growth Curves:

Characterization of intracellular bacterial growth was performed usingprimary bone marrow derived macrophages as previously described (Portnoyet al., 1989). Briefly, approximately, 8×10⁶ over night grown L.monocytogenes bacteria were used to infect 2×10⁶ macrophages cells,which resulted in infection of 1-2 bacteria per cell. Thirty minutesafter addition of bacteria macrophage monolayers were washed with PBSand media was added. At one h.p.i., gentamicin was added to 50 μg ml⁻¹,to limit the growth of extracellular bacteria. At 2, 4 and 6 hours postinfection 3 cover slips were taken and washed with water to lysemacrophages cells. Bacteria recovered from each coverslip were plated onBHI plates and the number of bacterial colonies was counted. FIG. 4shows that the LadR, VirR, and TetR mutant Listeria strains have anormal growth rate as compared to the wild type strain.

Sequencing of Tn917 Insertion Site

The locations of the Tn917 insertions were determined by plasmid rescue,as described in (Camilli et al., 1990). Briefly, chromosomal DNA from aTn917 insertion mutant was digested with XbaI, ligated at a DNAconcentration of 5 μg/mL, and used to transform DH5α cells and plated onkanamycin containing agar plates. Plasmid DNA was isolated fromtransformants and sequenced using a primer for Tn917:CAATAGAGAGATGTCACCG (SEQ ID NO:05) (FIGS. 5 and 6).

Listeria monocytogenes DNA Arrays

Oligos for L. monocytogenes arrays were synthesized by the institute forgenomic research (TIGR). The arrays were printed at the UCSF Center forAdvanced Technology. W.t. L. monocytogenes, lmo2589:: Tn917, lmo1408::Tn917, and lmo1745:: Tn917 mid-log cultures were filtered and frozen inliquid nitrogen. Bacteria were washed off the filter, and bacterial RNAwas isolated using phenol-chloroform extraction. Bacterial RNA wasamplified using Ambion MessageAmp™ II Bacteria Prokaryotic RNA Kit.Microarrays were gridded using Genepix and SpotReader, and analyzedusing Acuity. Genes that showed a 2 fold or greater difference from wildtype gene expression were selected for further analysis. Each sample wasdone in duplicate.

Example 2 Listeria monocytogenes Multidrug Resistance TransportersActivate a Cytosolic Surveillance Pathway of Innate Immunity

To address the question of how L. monocytogenes activates the hostcytosolic surveillance system, a L. monocytogenes Tn917 transposonlibrary (Camilli et al. J Bacteriol 172, 3738-44 (1990)) was screenedfor mutants that exhibited an enhanced or diminished Type I IFN responseupon infection of macrophages. Approximately 5000 L. monocytogenes Tn917mutants were used to infect bone marrow derived macrophages (BMM) in 96well plates. The amount of Type I interferon (i.e. IFN-β and/or IFN-α)secreted by macrophages during infection was measured by transferringmacrophage culture supernatant onto a Type I interferon reporter cellline, that produces luciferase in response to Type I interferon (Jianget al. Nat Immunol 6, 565-70 (2005)). 14 mutants that induced alteredinduction of Type I interferon in comparison to w.t. bacteria wereidentified. Among these, three mutants behaved like w.t. in theirability to infect, escape from the vacuole, and to grow insidemacrophages (FIG. 7 a). Transposon insertions in these mutants werelocated in genes encoding predicted transcription regulators (FIG. 7 b):ladR, previously shown to be a negative regulator of its adjacentmultidrug resistance transporter, MdrL (Huillet et al. FEMS MicrobiolLett 254, 87-94 (2006)), lmo2589 encoding a TetR-like protein andlmo1618 encoding a MarR-like protein (Grkovic et al. Proc Natl Acad SciUSA 98, 12712-7 (2001)). Real-time qRT-PCR analysis of IFN-β inductionin macrophages infected with these mutants confirmed that the ladRmutant induced 3-fold more IFN-β, the tetR mutant induced 20-fold moreIFN-β, and the marR mutant induced 3-fold less IFN-β compared to thelevel of IFN-β induced by w.t. bacteria (FIG. 7 c). While the 3 mutantsaffected the level of IFN-β in macrophages, none of them inducedmacrophage cell death as shown by a lactate dehydrogenase (LDH) releaseassay (FIG. 7 d). Interestingly, the LD₅₀ of ladR, tetR and marR mutantswere similar to w.t. L. monocytogenes (data not shown). Since thesemutants had the same LD₅₀ and grew like w.t. in macrophages, thesemutants would have been missed in screens that rely on intracellulargrowth or virulence such as signature tag mutagenesis (STM) ortransposon site hybridization (TraSH) (Sassetti et al. Proc Natl AcadSci USA 98, 12712-7 (2001); Hensel et al. Science 269, 400-3 (1995)).

This is the first description of the tetR and marR genes in L.monocytogenes. Interestingly, like the ladR transcription regulator, thetetR and marR regulators are located adjacent to putative multidrugresistance transporters of the major facilitator superfamily, named heremdrT and mdrM respectively (lmo2588, lmo1617) (FIG. 7 b). Among the 3MDRs, MdrM and MdrT are highly similar (46% amino-acid identity and 64%similarity) and share extensive similarity with the well-studiedmultidrug efflux transporter system, QacA-QacR, of Staphylococcus aureus(Grkovic et al. Proc Natl Acad Sci USA 98, 12712-7 (2001)). In S.aureus, QacR represses expression of the MDR qacA. In order to study theregulation of mdrL, mdrT, and mdrM expression by their adjacentregulators and their effect on the cytosolic innate immune response, aseries of in-frame deletions (Camilli et al. Mol Microbiol 8, 143-57(1993)) of the regulator genes, the MDR genes, and a double deletion ofeach MDR-regulator set of genes was generated (Table 2), with theexception of the tetR gene, for which the original transposontetR::Tn917 mutant was used. Table 2 lists the Listeria monocytogenesstrains used in this study. Listed for each strain is the relative levelof IFN-β induced by host macrophages, compared to the level of IFN-βinduced by w.t. L. monocytogenes.

TABLE 2 L. monocytogenes IFN-β induction/w.t. Strain Description L.monocytogenes 10403S Wild type 1 DP-L5396 ladR::Tn917 3 DP-L5398marR::Tn917 0.3 DP-L5397 tetR::Tn917 20 DP-L5449 Wt 10403S + pLIV2:mdrT3.5 DP-L5441 ladR− 3 DP-L5442 marR− 6 DP-L5443 mdrM− 0.3 DP-L5448mdrM− + pLIV2:mdrM 1 DP-L5444 mdrT− 1 DP-L5445 mdrL− 1 DP-L5446marR−/mdrM− 0.3 DP-L5447 ladR−/mdrL− 3

The expression level of each MDR was analyzed in bacteria grown in brothby real-time qRT-PCR. The results indicated that w.t. L. monocytogenesdid not express mdrL or mdrT, but expressed a measurable level of mdrM(FIG. 7 e). In the ladR-mutant the multidrug transporter mdrL was highlyinduced (˜30 fold) (Huillet et al. FEMS Microbiol Lett 254, 87-94(2006)). In addition, mutation in the ladR gene resulted in ˜3 foldinduction of mdrM, compared to its basal level of expression (FIG. 7 e).In the tetR::Tn917 mutant the adjacent multidrug transporter, mdrT, wasspecifically and highly induced (˜100 fold) (FIG. 7 e). In the case ofthe marR-regulator, the mdrM gene was located downstream of marR, andboth genes were predicted to be part of an operon (FIG. 7 b). While mdrMwas not expressed in the original marR::Tn917 mutant (not shown), it washighly induced in the marR in-frame deletion (˜70 fold) (FIG. 7 e),indicating that the transposon insertion blocked the expression of bothgenes due to polarity. These results clearly demonstrate that LadR, TetRand MarR negatively regulate the putative MDRs MdrL, MdrT and MdrM,respectively.

One common property of MDRs is that their expression is often induced bythe presence of their cognate drug substrates. For example, in theQacA-QacR system, the repression of gacA imposed by QacR is relievedwhen QacR binds toxic drugs, leading to induction of qacA expression(Grkovic et al. Proc Natl Acad Sci U S A 98, 12712-7 (2001); Schumacheret al. Mol Microbiol 45, 885-93 (2002)). When w.t. L. monocytogenes wasgrown in the presence of the commonly used toxic drugs,tetraphenylphosphonium (TPP) or rhodamine 6G (R6G) (Grkovic et al.,ibid), the transcription of all 3 MDRs were highly induced (FIG. 7 f),indicating that the regulator genes identified in this screen areinvolved in the regulation of MDR transporters.

Prior to this disclosure, nothing was known about the regulation of MDRtransporters during L. monocytogenes infection. For example, it was notclear whether the bacteria encounter toxic compounds during infectionthat might lead to induction of the MDRs. When macrophages were infectedwith strains deleted for each of the MDR transporters (mdrL, mdrT andmdrM) all 3 mutants were able to infect and replicate within macrophageslike w.t. bacteria (FIG. 8 a). In order to evaluate role(s) of theseMDRs in the induction of type I interferon, macrophages were infectedwith w.t. L. monocytogenes and the MDR mutants, and the induction ofIFN-β was analyzed at 4 hours post infection by real-time qRT-PCR. Theresults clearly demonstrated that among the 3 MDRs, MdrM was the onlyone necessary for induction of IFN-β, as this mutant induced only athird of the IFN-β induced by w.t. bacteria (FIG. 8 b). This result isconsistent with the observation that only mdrM exhibits basal expressionin w.t. L. monocytogenes (FIG. 7 e). Complementation of mdrM expressionwith an IPTG-inducible expression system (pLIV2 integration vector)(Fischetti et al. Gram-positive pathogens. ASM Press, Washington, D.C.,(2006)) restored the induction of IFN-β to the level induced by w.t.bacteria (FIG. 8 b). The marR deletion mutant, which overexpressed mdrM(FIG. 7 e), induced 6-fold more IFN-β than w.t. bacteria (FIG. 8 c).This level of IFN-β induction was completely dependent on mdrMexpression since it was not observed with the marR-mdrM double deletionmutant, which induced the same level of IFN-β as the mdrM mutant alone,thereby excluding a potential role for other MarR inducible genes (FIG.8 c). Further support for the role of MdrM in IFN-β induction came frominfecting macrophages with the ladR mutant. As shown in FIG. 7 e, LadRalso repressed the expression of mdrM, to a lesser extent than theMdrM-repressor. Infecting macrophages with the ladR-mutant resulted in a3 fold higher induction of IFN-β than with w.t. bacteria; however,infection with the double deletion ladR-mdrL mutant still induced 3 foldmore IFN-β then w.t. bacteria, indicating that this induction was notdue to over-expression of mdrL (FIG. 8 c). Microarray analysis comparingtotal gene expression of w.t. bacteria versus the ladR mutant revealedthat, besides mdrL, mdrM was the most differentially expressed gene inthe ladR-mutant (Table 3). Table 3 shows the results of microarrayanalysis comparing total gene expression of w.t. vs. ladR::Tn917 L.monocytogenes. RNA was isolated from bacteria grown to mid-log culturein Brain Heart Infusion media. All microarrays were done in duplicate,and the values shown are the average of 2 arrays, divided by wild typebacteria. All genes that showed a 2 fold difference vs. wild typebacteria are presented. Since mdrM over-expression in the marR-mutantresulted in enhanced host IFN-β expression (FIG. 7 e, 8 c), theinduction of IFN-β by the ladR-mutant was due to over-expression of mdrMand not mdrL. Overall, these results demonstrated a direct role for MdrMin activation of IFN-β in response to L. monocytogenes infection.Interestingly, w.t bacteria expressing IPTG-inducible MdrT (MdrMhomolog) also resulted in increased induction of IFN-β in infectedmacrophages (FIG. 8 d) (Fischetti et al., ibid). These observationsindicate that the induction of IFN-β was not restricted to MdrM, butcould be recapitulated by expression of homologous MDRs, likely withsimilar substrate specificity.

TABLE 3 Gene number Fold Expression/Wt lmo 1409 (mdrL) 164.3 lmo 1617(mdrM) 3.26 lmo 2434 3.21 hypothetical protein (similar to glutamatedecarboxylase) lmo 1069 3.16 hypothetical protein (similar to B.subtilis YlaI protein) lmo 2443 2.30 hypothetical protein lmo 2840 2.18hypothetical protein (similar to Sucrose phosphorylase) lmo 1308 0.39hypothetical protein (similar to arginine N-methyl- transferases)

To determine whether the lethality of the ladR, tetR and mdrM (pump1617)mutant strains was distinguishable from that of the wild type L.monocytogenes, the 50% lethality dose (LD₅₀) for the three strains weredetermined in Balb/c mice. Table 4 shows the results, which indicatethat the LD₅₀ of the mutant strains are similar to those of the wildtype.

TABLE 4 Strain Study AS07-023 Study AS07-029 WT  <7 × 10³ 1.39 × 10⁴ladR 6.90 × 10³ 1.27 × 10⁴ tetR 1.40 × 10⁴ 1.54 × 10⁴ Pump1617 (mdrM)2.34 × 10⁴  >8 × 10⁴

To study the biodistribution of the ladR, tetR and mdrM mutant strains,52 hour as well as 10-day time courses examining liver and spleencolonization in Balb/c mice were conducted as described previously byAuerbuch, V. et al. Infect. Immun. 69:5953-5957. FIG. 12 shows theresults of the 52 hour time course, which indicate that tetR andpump1617(mdrM) mutants are slightly attenuated at 24 h in the liver and52 h in the spleen.

Prior to this disclosure, how immune cells recognize intracellularpathogens such as L. monocytogenes was not fully understood. Thecytosolic innate immune response to L. monocytogenes is generallydescribed as independent of Toll-like receptors (TLRs) and theirsignaling adaptors, Myd88 and Trif, and dependent on the interferonregulatory factor 3 (IRF-3) (Perry et al. Cell Res 15, 407-22 (2005)).In order to test whether induction of IFN-β by the mutants identified inthis screen activated the same pathway, Myd88-Trif double knockoutmacrophages and IRF-3 deficient (O'Connell et al. J Exp Med 200, 437-45(2004)) macrophages were infected with ladR, marR, and tetR mutants. Theresults indicated that the increased induction of IFN-β by these mutantswas almost entirely independent of TLRs but absolutely dependent onIRF-3 (FIG. 9). One well-characterized cytosolic pathway that leads toIRF-3 activation and IFN-β expression is dependent on the cytosolicreceptors RIG-I and MDA-5 and their adaptor, MAVS (Yoneyama, M. et al.Nat Immunol 5, 730-7 (2004); Andrejeva, J. et al. Proc Natl Acad Sci USA101, 17264-9 (2004); Sun, Q. et al., ibid). We infected MAVS deficientmacrophages with w.t. L. monocytogenes, marR, and tetR::Tn917 mutants,and the induction of IFN-β by w.t. L. monocytogenes and the mutants wasindependent of MAVS (Sun, Q. et al., ibid; Soulat, D., et al.Cytoplasmic Listeria monocytogenes stimulates IFN-beta synthesis withoutrequiring the adapter protein MAVS. FEBS Lett 580, 2341-2346 (2006)).These results are consistent with the hypothesis that w.t. L.monocytogenes and the mutants induced altered levels of activation ofthe same host cytosolic surveillance pathway. To gain further insightinto the host pathways and downstream genes activated by bacterial MDRs,we compared the macrophage response to infection with w.t. L.monocytogenes, mdrM- and tetR::Tn917 mutants using microarray analysis.We used Type I IFN Receptor minus (IFNαβR−/−) macrophages to avoid thecomplication of IFN-β autocrine signaling. Macrophages infected with themdrM-mutant, which induced a 3 fold lower host IFN-β response, hadaltered expression of only 16 genes (by SAM analysis), all of which werediminished compared to macrophages infected with w.t. L. monocytogenes.Macrophages infected with the tetR::Tn917 mutant, which induced a 20fold higher IFN-β response, had strongly increased induction (by SAM andat least 4 fold) of 13 genes, compared to macrophages infected with w.t.L. monocytogenes. Interestingly, the genes whose expression was affectedby mdrM- and tetR::Tn917 mutants largely overlapped and are presented inFIG. 4. Moreover, the vast majority of these genes were previouslyidentified as “cytosolic response genes” (i.e. genes that are inducedonly by w.t. L. monocytogenes in the cytosol) and included IFN-β, IL-6,CCL5, and CXCL10 (Leber, J. et al. Distinct TLR- and NLR-MediatedTranscriptional Responses to an Intracellular Pathogen. PLoS Pathogens,In press (2007)). These experiments provided further evidence thatbacterial MDR expression specifically controlled the magnitude of thehost cytosolic surveillance pathway, including the expression of severalinnate immune signaling components.

To test the role of the cytosolic surveillance pathway in L.monocytogenes pathogenesis, we infected mice with w.t. L. monocytogenes,mdrM-, marR-, and tetR::Tn917 mutants. Interestingly, mice infected withthe mutant that induced 20 times more IFN-β, tetR::Tn917, had 20-foldlower bacterial loads in the liver, while the other mutants had w.t.levels of bacteria (FIG. 11 a). While L. monocytogenes' virulence mayrequire basal activation of the cytosolic surveillance system (Auerbuchet al., ibid) our new data are more consistent with a model in which L.monocytogenes avoids recognition by the host innate immune surveillancesystem.

This disclosure is the first report to demonstrate a role for bacterialMDR transporters in the activation of a host immune response. L.monocytogenes strains were generated that vary by 60-fold in the amountof IFNβ induced in infected macrophages, due to their levels of MDRexpression (FIG. 9 b). Importantly, the induction of IFNβ in infectedanimals recapitulated the results observed in tissue culture (FIG. 11).Type I interferons have wide ranging effects on innate and adaptiveimmune responses, and are used to treat multiple sclerosis, hepatitis C,and some malignancies (Theofilopoulos et al. Annu Rev Immunol 23, 307-36(2005); Maher et al. Curr Med Chem 14, 1279-89 (2007)). The strainsgenerated in this study can be used to provide novel insight into therole of IFN-β in linking innate and adaptive immunity, as well as beinginstrumental in the development of adjuvants and vaccines, and of use innew therapeutics.

Methods Summary:

Bacterial genetic screen. A total of 5000 individual L. monocytogenesTn917-LTV3-transposon insertion mutants (Camilli et al. J Bacteriol 172,3738-44 (1990)) were grown on BHI media in 96 well plates over night at30° C. Bone marrow-derived macrophages from C57BL/6 mice were plated on96 well plates, 4×10⁴ cells/well, and infected with 2×10⁶ bacteria. 30minutes post infection, macrophages were washed and gentamicin was added(50 μg/ml) to prevent extracellular growth of bacteria. At 6 hours postinfection 100 μl of macrophage culture media was taken and frozen at−80. The amount of Type I IFN in the media was detected using a reportercell line, ISRE-L929 (Jiang et al. Nat Immunol 6, 565-70 (2005)).ISRE-L929 cells were grown in 96 well plates and incubated with 40 μl ofinfected macrophage culture media for 4 h. Then, cells were lysed andluciferase activity was detected using Bright Glow Assay (Promega,E-2620) and measured with a luminescence counter (VICTOR3,PerkinElmer®).

Determination of Type I Interferon Level in Mice Serum. Balb/C mice wereinjected intravenous with either HBSS alone or 1×10⁴ cfu of eachbacterial strain in 100 μl of HBSS. 24 h later, mice were sacrificed andblood was collected to serum separator tubes (BD 39 5956) by cardiacpuncture. Serum was aliquotted and frozen at −80° C. until it was usedto assay Type I interferon in the ISREL929 reporter cell line asdescribed above. Assays were performed in duplicate.

Infections and Analysis of Gene Expression in Macrophages. RNA wascollected from infected macrophages at 4 hours post infection, andinduction of IFN-β was analyzed by qRT-PCR, as described20.

Listeria monocytogenes Gene Expression. Expression of MDR genes by L.monocytogenes growing in BHI broth was analyzed using real time qRT-PCRanalysis (Herskovits et al. PloS Pathog 3, e51 (2007)). Level of geneexpression was normalized to the level of expression of rpoB gene. Totest for expression of MDR genes after treatment with toxic drugs,Tetraphenylphosphonium (50 μM, Sigma) or Rhodamine 6G (50 μM, Sigma)were added for 1 hour, then total bacteria RNA was extracted andanalyzed by qRT-PCR (Herskovits et al., ibid).

Bacterial Strains. The L. monocytogenes strains used were a wild-typestrain, 10403S; or a strain containing an in-frame deletion of the hlygene (LLO, DP-L2161) (Jones et al. Infect Immun 62, 5608-13 (1994)). Alldeletion mutants generated in this study are summarized in Table 3.

Source of Mice. C57BL/6 and Balb/C mice were obtained from The JacksonLaboratory and Charles River respectively. Unless indicated otherwiseall knockout mice used in this 11 study were on the C57BL/6 background.Femurs or mice were obtained from the following source: MyD88Trif−/−,from B. Beutler, The Scripps Research Institute, La Jolla; IRF3−/− fromG. Cheng, Department of Microbiology, Immunology and Molecular Genetics,University of California.

Bacterial Genetic Screen. L. monocytogenes Tn917-LTV3-transposoninsertion libraries were used for the screen (Camilli et al. J Bacteriol172, 3738-44 (1990)). A total of 5000 bacterial insertion mutants weregrown on BHI media in 96 well plates and were grown overnight at 30° C.without shaking. Bone marrow-derived macrophages from C57BL/6 mice wereplated on 96 well plates, 4×10⁴ cells/well and were infected, intriplicate, with 2×10⁶ bacteria. Half an hour post infection,macrophages were washed to remove extracellular bacteria and gentamicinwas added at a concentration of 50 μg/ml to prevent extracellular growthof bacteria. At 4 hours post infection 100 μl of macrophages culturemedia was taken and frozen at −80. The amount of Type I IFN secreted bymacrophages during bacterial infection was detected using the reportergene luciferase, cloned under the regulation of Type I interferonsignaling pathway in L929 cell line, ISRE-L929 (Jiang et al., ibid.).ISRE-L929 cells were grown in 96 well plates and were incubated with 40μl of infected macrophages culture media for 4 h. Then, cells were lysedand luciferase activity was detected using Bright Glow Assay (Promega,E-2620) and light emission measurement by luminescence counter (VICTOR3,PerkinElmer®). Screening of mutants was done in triplicate for eachmutant on the same plate and 8 replicates of w.t. L. monocytogenes andhly-minus mutant as controls.

Generation of in-Frame Deletion Mutants in L. monocytogenes. In-framedeletions of L. monocytogenes genes were generated using splice-overlapextension (SOE)-PCR and allelic exchange, as previously described byCamilli et al (Camilli et al. Mol Microbiol 8, 143-57 (1993)). LD50study. Pathogenicity was determined in 5-6 week old BALB/C mice byserial twofold dilutions of each bacterial strain. LD50 calculationswere done by the method of Reed and Muench (Reed et al. The AmericanJournal of Hygiene 27, 493-497 (1938)).

Determination of Type I Interferon Level in Mice Serum. BALB/C mice wereinjected intravenously with either HBSS alone or 1×10⁴ cfu of eachbacterial strain in 100 μl of HBSS. Titers were confirmed by platinginjection stocks for cfu counting. 24 h later, mice were sacrificed andblood, collected by cardiac puncture, was placed in serum separatortubes (BD 39 5956) and processed per manufacturer's recommendations.Serum was aliquotted and frozen at −80° C. until Type I interferon levelcould be assayed. 50 μl of serum was thawed and used to assay Type Iinterferon in the ISRE-L929 reporter cell line as described for thebacterial genetic screen (above). Assays were performed in duplicate.

Bacterial Intracellular Growth Curves. Characterization of intracellularbacterial growth was performed using primary cultures of bone marrowderived macrophages as described (Portnoy et al. J Exp Med 167, 1459-71(1988)). Briefly, 2×10⁶ macrophages were infected with 4×10⁵ L.monocytogenes from an overnight culture. Thirty minutes after additionof bacteria macrophage monolayers were washed with PBS. At one h.p.i.,gentamicin was added to 50 μml⁻¹, to limit the growth of extracellularbacteria. At different time points post infection 3 cover slips weretaken and washed with water to lyse macrophages cells. Bacteriarecovered from each coverslip were plated on BHI plates and the numberof bacterial colonies was counted.

Infections and Analysis of Gene Expression in Macrophages. Approximately8×10⁶ L. monocytogenes were used to infect 2×10⁶ macrophages cellsseeded on 60 mm Petri dish. These numbers resulted in infection of 1-2bacteria per cell. Thirty minutes after addition of bacteria, macrophagemonolayers were washed three times with PBS and fresh media was added.At one hour post infection (h.p.i.), gentamicin was added to 50 μg ml⁻¹,to limit the growth of extracellular bacteria. Unless indicatedotherwise, RNA was collected at 4 h.p.i for further analysis. Inductionof IFN-β by macrophages analyzed by real time qRT-PCR as described(Herskovits et al., ibid.).

Listeria monocytogenes Gene Expression. Expression of MDR genes by L.monocytogenes grown in BHI broth was analyzed using real time qRT-PCRanalysis (Herskovits et al., ibid). Level of gene expression wasnormalized to the level of expression of rpoB gene in L. monocytogenes.Primers sequence: rpoB F (SEQ ID NO: 6): gcggatgaagaggataattacg; rpoB R(SEQ ID NO: 7): ggaatccatagatggaccgtta; mdrL F (SEQ ID NO: 8):ggganatggataacagaggc; mdrL R (SEQ ID NO: 9): gagcattgtcatcgcgg; mdrM F(SEQ ID NO: 10): ggtattttgattgttatgcttatgg; mdrM R (SEQ ID NO: 11):ttgtaaatcgttcaattaaaaaggc; mdrT F (SEQ ID NO: 12): aatagtacagcagtagaacg;mdrT R (SEQ ID NO: 13): ctgtaatatgcaaatcatcc. To test for induction ofMDR expression in the presence of toxic drugs, an over-night culture ofw.t. L. monocytogenes was diluted to O.D.600 of 0.01 and grown tomid-log in BHI media at 37C shaking. Tetraphenylphosphonium (50 μM,Sigma) and Rhodamine 6G (50 μM, Sigma) were added for 1 hour and thanbacteria were harvested, and total bacteria RNA was extracted andanalyzed by qRT-PCR (Herskovits et al., ibid.).

Microarray Analysis of L. monocytogenes Gene Expression.Oligonucleotides for L. monocytogenes arrays were synthesized by theinstitute for genomic research (TIGR), and the arrays were printed atthe UCSF Center for Advanced Technology. Midlog cultures of w.t. L.monocytogenes and ladR::Tn917 mutants were filtered and frozen in liquidnitrogen. Bacteria were washed off the filter, and bacterial RNA wasisolated using phenol-chloroform extraction. Bacterial RNA was amplifiedusing MessageAmp™ II Bacteria Prokaryotic RNA Kit (Ambion). Microarrayswere gridded using Genepix and SpotReader, and analyzed using Acuitysoftware (Herskovits et al., ibid.). Genes that showed a 2-fold orgreater difference from w.t. gene expression were selected for furtheranalysis. Each sample was done in duplicate.

Microarray Analysis of w.t. L. monocytogenes, mdrM-, and tetR::Tn917Infected IFNαβR Deficient Macrophages. Microarray analysis of IFNαβR−/−macrophages infected with w.t. L. monocytogenes, mdrM-, or tetR::Tn917mutants was done as previously described (Leber, J. et al., DistinctTLR- and NLR-Mediated Transcriptional Responses to an IntracellularPathogen. PLoS Pathogens, In press (2007); Herskovits et al.,ibid.),with the following modifications: 1) Macrophages were infected atMOI of 1, and RNA was collected 4 hours post infection. 2) Afternormalizing all microarrays to the uninfected controls, SAM analysis wasperformed with two-class unpaired designs to identify genes that weredifferentially expressed in w.t L. monocytogenes infected versus mdrM-or tetR::Tn917 infected macrophages, with a false discovery rate of 10%.Those genes that were statistically changed in mdrM-infectedmacrophages, and those genes that were statistically changed and atleast 4 fold differentially expressed (from w.t. L. monocytogenes) intetR::Tn917 infected macrophages were selected for further analysis(FIG. 10). Accession numbers for the genes in FIG. 10 are as follows:NM_(—)010510 (IFNβ), NM_(—)008332 (IFIT2), NM_(—)010501 (IFIT3),NM_(—)020557 (TYK1), NM_(—)031168 (IL6), BC030067 (CXCL10), NM_(—)027835(MDA5), NM_(—)021384 (Rsad2), NM_(—)145209 (OASL1), NM_(—)011854(OASL2), NM_(—)153564 (Gbp5), NM_(—)018734 (Gbp3), NM_(—)015783 (ISG15),NM_(—)172648 (111205), NM_(—)148927 (Plekh4), NM_(—)008013 (Fgl2),NM_(—)153287 (Axud1), and NM_(—)133234 (Bbc3). The un-annotated gene isRIKEN cDNA 1190002H23.

Lactate Dehydrogenase (LDH) Release Assay. BMMs macrophages wereinfected with w.t. L. monocytogenes and mutants at a multiplicity ofinfection (MOI) of one in the absence of gentamicin. Three, five andseven hours post infection, supernatant sample was removed and assayedfor LDH activity. Reagents were purchased from Sigma. The numbersreported were calculated with the mean LDH release from three wells of asingle experiment and are representative of two independent experiments.The L. monocytogenes cytotoxic strain S44A LLO was used as a positivecontrol.

Example 3 Listeria monocytogenes Mutants in a Multidrug ResistanceTransporter Gene and Multidrug Resistance Transporter GeneTranscriptional Regulators Induce an Altered Type I Interferon Response

Human type I interferons (IFNs) bind to a specific cell surface receptorcomplex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 andIFNAR2 chains. Homologous molecules to type I IFNs are found in manyspecies, including most mammals, and some have been identified in birds,reptiles, amphibians and fish species (Schultz et al., Developmental andComparative Immunology, Volume 28, pages 499-508). IFN-α and IFN-β aresecreted by many cell types including lymphocytes (natural killer (NK)cells, B-cells and T-cells), macrophages, fibroblasts, endothelialcells, osteoblasts and others. They stimulate both macrophages and NKcells to elicit an anti-viral response, and are also active againsttumors.

Mice lacking the type I interferon receptor are resistant to infectionwith wild type L. monocytogenes, producing high levels of IL-12 in lieuof IFN-αβ (Auerbuch et al., ibid). In addition to MHC and costimulatorymolecule signaling, it has been documented that Type I IFNs provide athird signal to CD8+ T cells to stimulate effective clonal expansion andeffector function. As such, modulation of Type I IFNs finds use inmounting a more effective adaptive immune response in vaccine platforms.To determine whether ladR, tetR and mdrM (pump1617) mutant strainsinduced Type I IFN responses differed from that of the wild type L.monocytogenes, L929 mouse fibroblast line (ATCC Cat. No. CCL-1) cellswere exposed to serum from Balb/c mice infected with w.t., ladR, tetRand mdrM (pump1617) mutant strains. The results presented in FIG. 13indicate that tetR and ladR strains show elevated Type I IFN responsesrelative to w.t. L. monocytogenes.

To investigate the effects on secretion of other cytokines by infectionwith the ladR, tetR and mdrM (pump1617) mutant strains relative to w.t.,cytometric bead array (CBA) assays were performed as described by Chenet al. (Chen R, Lowe L, Wilson J D, Crowther E, Tzeggai K, Bishop J E,et al., Simultaneous quantification of six human cytokines in a singlesample using microparticle-based flow cytometric technology. Clin Chem1999; 45:1693-1694) using serum from infected Balb/c mice. FIG. 14 showsthe results, which indicate that MCP-1, IFN-γ, IL-12p70, TNF-α, IL-10,and IL-6 secretion is lower for all mutants.

To assess the effects of infection by ladR, tetR and mdrM (pump1617)mutant strains relative to w.t. with respect to NK cell maturation andactivation, hepatocytes from Balb/c mice were harvested 2 dayspost-infection, as schematically shown in FIG. 15. Single cellsuspensions were created, the cells were stained for known NK cellmarkers and flow cytometry performed on the labeled cells to assess NKmaturation. FIG. 16 shows the results, which indicate that all strains,mutant and w.t., yielded similar liver NK cell maturation profiles. NKcell activation was then assessed by quantitating the number ofCD11b+/DX5+NK cells, as shown in FIG. 17. The results indicate that mdrM(pump1617) infection results in less NK activation than other mutants,which are comparable to w.t.

Next, CD8+ T cell response to four L. monocytogenes epitopes wasassessed, as shown in FIGS. 18 and 19. MdrM (pump1617) shows a greaterL. monocytogenes epitope-specific CD8+ T cell response than ladR andw.t., which in turn is greater than that of tetR (FIG. 19). Similarly,CD4+ T cell specific epitopic response was reduced in the case of tetRand elevated relative to w.t. in the case of mdrM (pump1617).Accordingly, mutation of L. monocytogenes multidrug resistancetransporter genes and their transcriptional regulators induces analtered Type I interferon response in the host.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

That which is claimed is:
 1. A Listeria bacterium comprising a mutationwhich modulates the expression of a multidrug resistance transporter,wherein said Listeria bacterium modulates interferon-β production inmacrophages, wherein said mutation is a mutation in a transcriptionregulator gene chosen from a TetR gene, a LadR gene and a MarR gene or amutation in a multidrug resistance transporter gene chosen from an MdrTgene and an MdrM gene.
 2. The Listeria bacterium according to claim 1,wherein said mutation is a mutation in a transcription regulator gene.3. The Listeria bacterium according to claim 1, wherein said mutation isa mutation in a multidrug resistance transporter gene.
 4. The Listeriabacterium according to claim 2, wherein said transcription regulatorgene encodes a product that regulates a multi drug resistancetransporter.
 5. The Listeria bacterium according to claim 3, whereinsaid multidrug resistance transporter gene encodes a product thatactivates interferon-β production.
 6. The Listeria bacterium accordingto claim 1, wherein said mutation is chosen from an insertion and adeletion mutation.
 7. The Listeria bacterium according to claim 1,wherein said Listeria bacterium is Listeria monocytogenes.
 8. TheListeria bacterium according to claim 1, wherein said Listeria bacteriumis attenuated.
 9. The Listeria bacterium according to claim 1, whereinsaid Listeria bacterium increases interferon-β production in macrophagesas compared to a wild type Listeria bacterium.
 10. The Listeriabacterium according to claim 1, wherein said Listeria bacteriumdecreases interferon-β production in macrophages as compared to a wildtype Listeria bacterium.
 11. The Listeria bacterium according to claim9, wherein said increase in interferon-β production is Toll-likereceptor independent.
 12. The Listeria bacterium according to claim 9,wherein said increase in interferon-β production is IRF-3 dependent. 13.The Listeria bacterium according to claim 9, wherein said increase ininterferon-β production does not induce macrophage cell death.
 14. TheListeria bacterium according to claim 1, wherein said bacteriumcomprises a heterologous nucleic acid.
 15. The Listeria bacteriumaccording to claim 14, wherein said heterologous nucleic acid isintegrated.
 16. The Listeria bacterium according to claim 14, whereinsaid heterologous nucleic acid encodes at least one product.
 17. TheListeria bacterium according to claim 16, wherein said at least oneproduct is an antigen.
 18. The Listeria bacterium according to claim 1,further comprising a mutation in a UvrA gene and/or a UvrB gene.
 19. Avaccine comprising an attenuated Listeria bacterium having a mutationwhich modulates the expression of a multidrug resistance transporter,wherein said attenuated Listeria bacterium modulates interferon-βproduction in macrophages, wherein said mutation is a mutation in atranscription regulator gene chosen from a TetR gene, a LadR gene and aMarR gene or a mutation in a multidrug resistance transporter genechosen from an MdrT gene and an MdrM gene.
 20. The vaccine according toclaim 19, wherein said mutation is a mutation in a transcriptionregulator gene.
 21. The vaccine according to claim 19 wherein saidmutation is a mutation in a multidrug resistance transporter gene. 22.The vaccine according to claim 19, wherein said mutation is chosen froman insertion mutation and a deletion mutation.
 23. The vaccine accordingto claim 19, wherein said modulation of interferon-β production does notinduce macrophage cell death.
 24. The vaccine according to claim 19,wherein said Listeria bacterium is Listeria monocytogenes.
 25. Thevaccine according to claim 19, wherein said Listeria bacterium furthercomprises a mutation in a UvrA gene and/or a UvrB gene.